Neurodegenerative diseases are characterized by neuroinflammation, a symptom with growing interest directed towards the development of active drugs for the reduction or elimination of its negative effects. It is largely reported on the anti-inflammatory and their potential neuroprotective activity in some neural cells of Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to the class of ?-3 polyunsaturated fatty acids. Most of the observed biological activities of these fatty acids are maintained, and in some case enhanced, in the corresponding amide derivatives or oxygenated metabolites. While free acids EPA and DHA are commercially available, the corresponding amides were prepared by aminolysis of the ethyl esters of EPA and DHA with ethanolamine in the presence of immobilized lipase from Candida antarctica (Novozym 435) and molecular sieves in tert-butyl methyl ether. Our study aims to elucidate the protective effect of both EPA and DHA, as well as the corresponding N-ethanolamides EPA-EA and DHA-EA, on Olfactory Ensheathing Cells (OECs) exposed to lipopolysaccharide (LPS)-induced neuroinflammation for 24 h. OECs are glial cells located in the olfactory system, which is the first to show a deficit in neurodegenerative diseases. To verify the anti-inflammatory effect of these compounds on OEC cultures and on cell morphological features, the expression of some cytoskeletal proteins, such as Vimentin and Glial Fibrillary Acid Protein (GFAP), was evaluated by immunocytochemical procedures. In addition, MTT test was carried out to establish the non-toxic concentrations and the optimal time of exposure. Mitotoxicity and cytotoxicity levels in stressed were detected by using the HCS Mitochondrial Health Kit, while apoptosis was determined by staining with the TUNEL Alexa Fluor Imaging assay. Our results show a decrease of GFAP and Vimentin expression in OECs treated with EPA or DHA acids or EPA-EA or DHA-EA and stressed with LPS when compared with OECs exposed to LPS alone. While a protective role on cell morphology is predominantly observed for EPA and DHA, the amides EPA-EA and DHA-EA mainly show anti-inflammatory effects, superior to those of free acids. These results highlight that all the tested compounds have anti-inflammatory activity on LPS-exposed OECs and may provide an innovative tool to contrast neuroinflammation, which plays a key role in several neurodegenerative diseases.

Neuroinflammation is a common symptom in the onset of different neurodegenerative diseases and growing interest is directed towards the development of active drugs for the reduction or elimination of its negative effects. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to the class of ?-3 polyunsaturated fatty acids, have been largely investigated for their anti-inflammatory activity and their potential as neuroprotective agents has been evaluated on some neural cells1. Most of the observed biological activities of these fatty acids are maintained, and in some case enhanced, in the corresponding amide derivatives or oxygenated metabolites2. Our study aims to elucidate the protective effect of both EPA and DHA, as well as the corresponding N-ethanolamides EPA-EA and DHA-EA, on Olfactory Ensheathing Cells (OECs) exposed to lipopolysaccharide (LPS)-induced neuroinflammation. OECs are glial cells located in the olfactory system, which is the first to show a deficit in neurodegenerative diseases. To verify the anti-inflammatory effect of these compounds on OEC cultures and on cell morphological features, the expression of some cytoskeletal proteins, such as Vimentin and Glial Fibrillary Acid Protein (GFAP), was evaluated by immunocytochemical procedures. In addition, MTT test was carried out to establish the non-toxic concentrations and the optimal time of exposure. Our results show a decrease of GFAP and Vimentin expression in OECs treated with EPA or DHA acids or EPA-EA or DHA-EA and stressed with LPS when compared with OECs exposed to LPS alone. While a protective role on cell morphology is predominantly observed for EPA and DHA, the amides EPA-EA and DHA-EA mainly show anti-inflammatory effects, superior to those of free acids. These results highlight that all the tested compounds have anti-inflammatory activity on LPS-exposed OECs and may provide an innovative tool to contrast neuroinflammation, which plays a key role in several neurodegenerative diseases.

Called "man's most versatile chemical servant", by Lesser in 1949, (1) glycerol has lately become the main platform biochemical of the chemical industry (2). Lesser was mentioning 1583 commercial direct uses for glycerol due to its unique chemical, physical and biological properties: humectant or hygroscopic agent, vehicle, solvent, sweetening agent, emollient, lubricant, softening and demulcent agent, antifreeze, refrigerant, preservative and many other.

Background: A commonly used measure to reflect the intake of the long-chain omega-3 fatty acids EPA and DHA is the omega-3 index, defined as the sum of EPA + DHA as % of total fatty acids in erythrocyte membrane. When the omega-3 index changes it follows that the relative fractions of other fatty acids in the membrane are also changed. In the present study, increasing doses of a preparation of omega-3 rich phospholipids extracted from krill oil were administered orally to non-human primates for 12 weeks and the time course of EPA, DHA and 22 other fatty acids in erythrocytes was determined bi-weekly during treatment and for 8 weeks after cessation of treatment. Plasma concentrations of six endocannabinoid-type mediators being downstream metabolites of some fatty acids analyzed in erythrocytes were also determined.

Translationally Controlled Tumor Protein (TCTP) is a survival factor in tumor cells overexpressed in poorly differentiated breast cancer cells. TCTP is a specific target of Dihydroartemisinin (DHA). DHA is a metabolite of Artemisinin, the active principle of Artemisia annua L. DHA is an anti-malaria drug with antitumor properties. We studied the effect of DHA on human breast cancer cell lines (such as MDA-MB-231 and SKBR3 cells) with more aggressive phenotype. Our results show that DHA inhibits breast cancer cells growth and induces apoptosis by reducing the levels of the phosphorylated form of TCTP. We also show that DHA improves the antitumor effect of the conventional chemotherapy drugs, such as Doxorubicin and Trastuzumab. Altogether, these results suggest that phospho-TCTP is a novel therapeutic target for breast cancer cells. DHA in combination with conventional chemotherapeutics is a novel strategy to treat breast cancer.

Upregulation of Translationally Controlled Tumor Protein (TCTP) is associated with poorly differentiated aggressive tumors, including breast cancer, but the underlying mechanism(s) are still debated. Here, we show that in breast cancer cell lines TCTP is primarily localized in the nucleus, mostly in the phosphorylated form. The effects of Dihydroartemisinin (DHA), an anti-malaria agent that binds TCTP, were tested on breast cancer cells. DHA decreases cell proliferation and induces apoptotic cell death by targeting the phosphorylated form of TCTP. Remarkably, DHA enhances the anti-tumor effects of Doxorubicin in triple negative breast cancer cells resulting in an increased level of apoptosis. DHA also synergizes with Trastuzumab, used to treat HER2/neu positive breast cancers, to induce apoptosis of tumor cells. Finally, we present new clinical data that nuclear phospho-TCTP overexpression in primary breast cancer tissue is associated with high histological grade, increase expression of Ki-67 and with ER-negative breast cancer subtypes. Notably, phospho-TCTP expression levels increase in trastuzumab-resistant breast tumors, suggesting a possible role of phospho-TCTP as a new prognostic marker. In conclusion, the anti-tumor effect of DHA in vitro with conventional chemotherapeutics suggests a novel therapeutic strategy and identifies phospho-TCTP as a new promising target for advanced breast cancer.

CORSO DI LAUREA MAGISTRALE IN BIOTECNOLOGIE MEDICHE TESI DI LAUREA SPERIMENTALE

Upregulation of Translationally Controlled Tumor Protein (TCTP) is associated with poorly differentiated aggressive tumors, including breast cancer, but the underlying mechanism(s) are still debated. Here, we show that in breast cancer cell lines TCTP is primarily localized in the nucleus, mostly in the phosphorylated form. The effects of Dihydroartemisinin (DHA), an anti-malaria agent that binds TCTP, were tested on breast cancer cells. DHA decreases cell proliferation and induces apoptotic cell death by targeting the phosphorylated form of TCTP. Remarkably, DHA enhances the anti-tumor effects of Doxorubicin in triple negative breast cancer cells resulting in an increased level of apoptosis. DHA also synergizes with Trastuzumab, used to treat HER2/neu positive breast cancers, to induce apoptosis of tumor cells. Finally, we present new clinical data that nuclear phospho-TCTP overexpression in primary breast cancer tissue is associated with high histological grade, increase expression of Ki-67 and with ER-negative breast cancer subtypes. Notably, phospho- TCTP expression levels increase in trastuzumab-resistant breast tumors, suggesting a possible role of phospho-TCTP as a new prognostic marker. In conclusion, the anti-tumor effect of DHA in vitro with conventional chemotherapeutics suggests a novel therapeutic strategy and identifies phospho- TCTP as a new promising target for advanced breast cancer.

Microalgae offer potential for numerous commercial applications, including the production of w-3 polyunsaturated fatty acids (w-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These valuable fatty acids are important for a variety of nutraceutical and pharmaceutical purposes, and this has led to increased consumption as food supplements. Currently, the main source of EPA and DHA is marine fish oil, which is inadequate and unattractive mostly because of taste, odor and stability problems, and unsustainable because it contributes to overfishing global fish stocks. There is a variety of alternative EPA and DHA sources such as bacteria, fungi, and plants that are currently being explored for commercial production.Fungi require organic carbon sources, plants need arable land, have longer growth times and have no enzymatic activity for producing long chain PUFA, unless genetically modified. Several microalgal species that are the primary EPA and DHA producers in the marine food chain, contain high level of specific PUFAs and can grow under a variety of autotrophic, mixotrophic and heterotrophic conditions, offer a valuable and sustainable alternative source of PUFAs that may help to meet the increasing demand for fish oil. This presentation outlines an introductory overview of PUFA-related health benefits in humans, describes PUFA occurrence in different microalgal species together with the most important growth parameters affecting fatty acid content and profile and depicts the major pathways of their biosynthesis in selected species of microalgae. Furthermore, the recent improvements in cultivation systems utilised for large-scale microalgae mass production are illustrated and the feasibility of cultivating selected microalgal strains as a potential source of EPA evaluated. The opportunities and bottlenecks of microalgal production and refinery are also discussed.

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm2). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.

Among the components of the human diet, meat supplies a wide range of nutrients and is a source of fat both as adipose tissue and as marbling within the muscle. The most important characteristics of lipids are the quantity and type of fatty acid (saturated fatty acids and polyunsaturated fatty acids) and the cholesterol concentration. The balance in the diet between omega-3 PUFA formed by alpha-linolenic acid and omega-6 PUFA by linoleic acid, and the ratio is also a risk factor in cancer and coronary heart disease. Ratios less that 4 are strongly recommended. Different amounts of omega-3 fatty acids in protected form from rumen, do make the difference in meat quality of lamb and in particular in fatty acids compounds. Beneficial effect was found in the composition of the longissimus muscle of lamb fed with 7% of omega-3. Deeper studies on this particular topic will strongly affect prevention in medicine and clear pathology. The possibility of taking in protective compounds with food is very important for the human health and even more for diseased persons.

Negli ultimi anni, il consumatore si attende dagli alimenti specifiche caratteristiche quali valore nutrizionale, sanità, genuinità. Pertanto, le produzioni animali devono essere realizzate tenendo conto della salvaguardia dell'ambiente, del benessere animale e della salute umana. Negli allevamenti devono essere utilizzate tecniche per ridurre l'inquinamento dell'aria, dell'acqua e del suolo, mentre sull'animale le strategie adottate, soprattutto alimentari, sono finalizzate alla tracciabilità, sicurezza e livello qualitativo dei prodotti di origine animale. Tra questi ultimi la carne è una delle principali produzioni zootecniche e gioca un ruolo importante nell'alimentazione umana. Il consumo di carne rossa, comunque, ha subito un calo a causa del suo elevato contenuto in acidi grassi saturi che sono ritenuti dannosi per la salute umana. Alla luce del recente sviluppo del mercato dei cosiddetti " functional food" o alimenti funzionali, ricchi o "arricchiti" di componenti biologicamente attivi che sono in grado di ottimizzare il benessere fisico e mentale dell'uomo e di ridurre anche il rischio di contrarre malattie, sono stati condotti diversi studi allo scopo di modificare la composizione acidica del grasso intramuscolare della carne e rendere quindi tale prodotto un alimento "funzionale".

Si è studiato l'effetto di diete arricchite in acido docosaesaenoico (DHA-22:6n-3) e/o vitamina E (vE) sulla qualità e sui parametri biochimici del materiale seminale di maschi riproduttori di coniglio. Sono stati utilizzati 52 animali suddivisi in 4 gruppi secondo il seguente schema: A: dieta controllo; B: dieta A + 200 mg vE /kg; C: dieta A + 1,5% di olio di pesce (FO); D: dieta A + 200 mg vE/kg + 1,5% FO. Sul materiale seminale si sono eseguite periodicamente le seguenti valutazioni qualitative: volume, concentrazione, motilità e vitalità. Si è inoltre analizzata la composizione lipidica degli spermatozoi. I lipidi sono stati separati nelle diverse classi: fosfolipidi, colesterolo libero, acidi grassi liberi e trigliceridi; dopo la separazione è stata determinata la composizione acidica mediante Gas Cromatografia. I fosfolipidi sono risultati la principale classe presente negli spermatozoi, seguita dal colesterolo libero. I fosfolipidi hanno evidenziato un elevato contenuto di acido docosapentaenoico (DPA-22:5n-6) (circa il 32% degli acidi grassi totali) e un basso contenuto di 22:6n-3 (0.7%) nei gruppi A e B. Il livello di DHA nei gruppi C e D è aumentato di circa 7 volte rispetto ai gruppi A e B (circa 5% contro 0,7%). Contemporaneamente si è avuta una significativa diminuzione del 22:5n-6. Anche il livello di vE nella dieta ha influito sui valori di n-3 e n-6, con un inatteso effetto negativo sul contenuto di acidi grassi polinsaturi (PUFA). Sono necessarie ulteriori ricerche per comprendere il ruolo dei PUFA a lunga catena sulla funzionalità spermatica nella specie cunicola.

Si è studiato l'effetto di diete arricchite in acido docosaesaenoico (DHA-22:6n-3) e/o vitamina E (vE) sulla qualità e sui parametri biochimici del materiale seminale di maschi riproduttori di coniglio. Sono stati utilizzati 52 animali suddivisi in 4 gruppi secondo il seguente schema: A: dieta controllo; B: dieta A + 200 mg vE /kg; C: dieta A + 1,5% di olio di pesce (FO); D: dieta A + 200 mg vE/kg + 1,5% FO. Sul materiale seminale si sono eseguite periodicamente le seguenti valutazioni qualitative: volume, concentrazione, motilità e vitalità. Si è inoltre analizzata la composizione lipidica degli spermatozoi. I lipidi sono stati separati nelle diverse classi: fosfolipidi, colesterolo libero, acidi grassi liberi e trigliceridi; dopo la separazione è stata determinata la composizione acidica mediante Gas Cromatografia. I fosfolipidi sono risultati la principale classe presente negli spermatozoi, seguita dal colesterolo libero. I fosfolipidi hanno evidenziato un elevato contenuto di acido docosapentaenoico (DPA-22:5n-6) (circa il 32% degli acidi grassi totali) e un basso contenuto di 22:6n-3 (0.7%) nei gruppi A e B. Il livello di DHA nei gruppi C e D è aumentato di circa 7 volte rispetto ai gruppi A e B (circa 5% contro 0,7%). Contemporaneamente si è avuta una significativa diminuzione del 22:5n-6. Anche il livello di vE nella dieta ha influito sui valori di n-3 e n-6, con un inatteso effetto negativo sul contenuto di acidi grassi polinsaturi (PUFA). Sono necessarie ulteriori ricerche per comprendere il ruolo dei PUFA a lunga catena sulla funzionalità spermatica nella specie cunicola.